Supplementary Material

Involvement of ribosomal protein L6 in assembly of functional 50S ribosomal

subunit in Escherichia coli cells

Yuta Shigeno a, Toshio Uchiumi b, and Takaomi Nomura a*

a Division of Applied Biology, Faculty of Textile Science and Technology, Shinshu University,

Ueda 386-8567, Japan

b Department of Biology, Faculty of Science, Niigata University, Niigata 950-2181, Japan

* Corresponding author. E-mail address: nomurat@shinshu-u.ac.jp

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Table S1.

All strains, plasmids and primers used in this study

Strains or plasmids or Primers Genotype or charactaristic or Seaquancing

Strains

MG1655 F– λ– ilvG– rfb-50 rph-1

Red MG1655 red (TcR) hsdR:Ap [25]

Red-∆L6 MG1655 red (TcR) hsdR:ApR rplF::cat transformed with comlementing plasmid pJKspcL6

∆L6 mutant MG1655 rplF::cat transformed with comlementing plasmid pBADL6

Plasmids

pJKsocL6 Ligated fragment of rplF under the spc promoter cloned into the plasmid pJK289 [23],

which was low-copy-number (1–2 copies per cell) and conferred resistance to kanamycin pBADL6 rplF cloned into the plasmid pBAD24 [24], which was carrying arabinose-inducible rplF

and conferred resistance to ampicillin pBADHisL6 N-terminal His-tagged L6 fusion gene cloned into the plasmid pBAD24, which was

constructed by inverse-PCR method using the plasmid pBADL6 as a templatePrimers (5' → 3')

1 GCGCGGATCCTTACTTCTTCTTAGCCTCTT

2 GGCGAAATTATCTGCTACGT

3 TTTTTTCCTCCGATTAGGCTACGTAGCAGATAATTTCGCCTTTAGTGCTCCGCTAATGTC

4 GCGCGAATTCCTCTGCAAGGTCGCGTTGTT

Construction of plasmid pBADL6

5 CTCCATACCCGTTTTTTTGGGCTAGCAGGAGGAATTCACCATGTCTCGTGTTGCTAAAGC

6 CATAACATCAAACATCGACCCACGGCGTAACGCGAAGCTTTTACTTCTTCTTAGCCTCTT

Construction of plasmid pBADHisL6

7 ATGCATCATCATCATCATCATAGCAGCGGCTCTAGAATGTCTCGTGTTGCTAAAG

8 GGTGAATTCCTCCTGCTAGCCC

9 (a) CTTACTCTGAAGTATTTCCAGGG

10 TTTTTCCTCCGATTAGGCTA

11 (b) TGGCGAAATTATCTGCTACGTAGCCTAATCGGAGGAAAAAATGGAGAAAAAAATCACTGG

12 (c) GCACGACTTCGTCGGCGTAACGAACACCCTTGCCTTTATACACTTATTCAGGCGTAGCA

13 TATAAAGGCAAGGGTGTTCG

14 GTGAGCCATCTTACACCTCT

PCR analysis

a (9) CTTACTCTGAAGTATTTCCAGGG

b (11) TGGCGAAATTATCTGCTACGTAGCCTAATCGGAGGAAAAAATGGAGAAAAAAATCACTGG

c (12) GCACGACTTCGTCGGCGTAACGAACACCCTTGCCTTTATACACTTATTCAGGCGTAGCA

d CCAACTACAGTCAGAGCTGTG

　 e TGCAGATAATACCCTGACCTTCGGTCCGCG

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Fig. S1. Polymerase chain reaction (PCR) and sequencing screening of a homologous

recombinant. (A) Design of primers to analyze for the desired homologous recombination. Black

arrows represent primers used in this analysis. (B) Agarose gel electrophoresis of PCR products

obtained with primer pairs presented in (A); M, ∆L6 mutant; P, MG1655 strain (control). (C)

Nucleotide sequence near rplF in the ∆L6 mutant: Insert region, italic black; cat, bold italic

black; a portion of rplF, underlined light gray; other regions, gray.

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Fig. S2. Ribosomal protein components of the 30S subunits from each growth phase. Samples

of the 30S subunit peaks from wild-type ribosomes (lane 1) and ∆L6 mutant ribosomes obtained

at LI-phase (lane 2), S-phase (lane 3), and LII-phase (lane 4) (Fig. 3D) were analyzed by 16.5%

(w/v) SDS-PAGE and the gel was stained with silver.

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Fig. S3. Arabinose-induced L6 expression in Escherichia coli cells. The plasmid pBADHisL6,

which directs the expression of the N-terminally His-tagged ribosomal protein L6, was

constructed using inverse-polymerase chain reaction (PCR) with primer pair 13/14 and the

plasmid pBADL6 as a template. Wild-type E. coli strain MG1655 transformed with plasmid

pBADHisL6 was cultivated in liquid LB medium containing 50 µg/ml ampicillin until the OD600

reached 0.5. L-arabinose was added to a final concentration of 65 mM and the cells were

cultured for an additional hour to express His-tagged L6. The same experiment was performed in

the absence of arabinose, except that additional cultivation was carried out for 0, 1, 2, 3, and 5 h.

The harvested cells were analyzed by 16.5% (w/v) SDS-PAGE, and the proteins separated on the

gel were transferred to a PVDF membrane (Immobilon-P, Merck-Millipore, USA). The His-

tagged proteins were detected by using Immobilon Western Chemiluminescent HRP Substrate

(Merck-Millipore, USA) with HisProbe-HRP (Thermo Fisher Scientific, USA).

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